IDENTIFICATION AND CHARACTERIZATION OF A NITRATE REDUCTASE GENE FROM BEAN (PHASEOLUS-VULGARIS) CONTAINING 4 INTRONS

A structural gene encoding nitrate reductase (NR) in bean (Phaseolus v ulgaris) has been cloned and sequenced. The NR gene encodes a protein of 890 amino acids with a molecular mass of 100 kDa. Comparison to the other known NR gene from bean reveals 76% amino acid identity and com parison to NRs from other species shows amino acid identities ranging from 67 to 77%. At three positions the amino acid sequence displays di fferences from residues conserved in all other known NR proteins. The coding sequence is interrupted by four introns. Three of them are loca ted at conserved positions in the region encoding the molybdenum cofac tor-binding domain. The fourth intron is located in the hinge region b etween the heme and the FAD domain. This is the only example in which more than three introns have been found in a higher plant NR gene. The mRNA cap site was identified as an adenosine 79 nucleotides (nt) upst ream of the ATG translation star? codon. Northern analysis shows that the gene is nitrate inducible and highly expressed in trifoliolate lea ves of 2D-day-old bean plants and only weakly expressed in roots. The gene is also induced by light and sucrose in leaves of dark-adapted pl ants. The mRNA displays diurnal oscillation under the control of a cir cadian rhythm. Putative conserved GATA motifs in the promoter are disc ussed.